Spectrophotometric measurement of hemoglobin concentration of a blood sample has been used for decades on hematology analyzers. Typically, a blood sample is exposed to a lytic reagent to lyse the red blood cells, and then the released hemoglobin molecules in the sample mixture form a chromogen with a ligand or stabilizer in the reagent. A flow of the sample mixture is delivered through a cuvette in the analyzer, and absorption of the sample mixture is measured and used to calculate hemoglobin concentration of the blood sample.
In the existing hematology analyzers, commonly the measurement window of the cuvette includes two opposing parallel walls disposed along or in parallel with the flow path. A light source and a detector are disposed on opposing sides of the window for measuring absorption of the sample mixture passing through the cuvette. The optical length, namely the distance between the inner side of the two walls of the cuvette, is typically about 10 mm to ensure a sufficient amount of sample for measurement. A known problem in the existing measurement is attachment of micro-bubbles in the sample mixture to the walls of the cuvette, which causes errors in the measurement. Commonly, a large volume of a cleaning solution is used on the analyzers after analysis of each sample to remove and prevent accumulation of micro-bubbles on the surface of the cuvette. However, when the dimensions of the flow path in the cuvette decrease, the ratio of surface area to the volume of the sample mixture increases, and prevention of micro-bubble attachment becomes substantially more difficult.
On the other hand, most automated hematology analyzers have multiple reagents and cleaning solution on board for automated sample preparation and measurements on the instruments. Although these hematology analyzers have known advantages of high capacity, batch sample analysis, automated sample aspiration and preparation, and automated cleaning cycles, these instruments are relatively high cost and use large quantity of reagents, which requires high maintenance of the instruments and reagent inventory management. Therefore, it is difficult to adapt these instruments in a near-patient test environment, such as in the emergency room, where a minimum sample volume and minimum reagent maintenance are required. In near-patient tests, a small volume of blood sample may be collected from fingerstick, and is used to obtain a complete blood count (CBC) on an analyzer. As such, substantially reduced reagent volumes are used to maintain desired cell concentrations to ensure accurate measurement.
In the recent years, disposable cassette containing reagents for analysis of one sample and blood analyzers adapted to use the disposable cassettes have been developed for meeting such a need in near-patient testing. The disposable cassette contains pre-filled reagents with predetermined volumes designated for a complete analysis of one sample. To accommodate for the small sample and reagent volumes, the fluid volume in the cuvette used for hemoglobin measurement on a hematology analyzer needs to be reduced. This significantly increases the ratio of surface area of the flow path to the volume of the sample mixture, and renders extremely difficult to control micro-bubble formation and attachment within the cuvette when the sample mixture passes through and when it is measured in the cuvette. Moreover, it is also technically challenging to remove micro-bubbles attached to or accumulated in the cuvette using a small volume of a cleaning solution provided in the cassette only.
Therefore, there exists a clear need for a spectrophotometer using a cuvette with a minimum fluid volume for providing an accurate measurement of hemoglobin concentration of a blood sample and meeting requirements of in-vitro clinical diagnosis. Furthermore, it is strongly desirable to provide a miniature cuvette that can effectively minimize micro-bubble formation and accumulation within the cuvette and minimize reliance on the amount of cleaning solution for removal of the micro-bubbles.